An avrcap1b nanobody and uses thereof

Abstract

The present application relates to an AVRcap1b nanobody and application thereof, and belongs to the field of biological medicine, wherein the present application develops an anti-AVRcap1b nanobody through multiple steps such as detection and screening by using a yeast two-hybrid screening system, the screening cost is greatly reduced compared with traditional antibodies, the research and development cycle is shorter, the requirements for experimental environment and equipment are simpler, and the applicability is wide. Compared with traditional AVRcap1b antibodies, the developed AVRcap1b nanobody has smaller molecular weight, better tissue permeability, is more stable, and has low immunogenicity. In summary, the AVRcap1b nanobody has outstanding advantages in terms of antibody affinity and targeting specificity, small molecule size, stability and transformability, production cost and the like.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of biological medicine, and relates to an AVRcap1b nanobody obtained by yeast two-hybrid screening and application thereof. BACKGROUND

[0002] AVRcap1b is an effector protein secreted by Oomycetes, and its target is a membrane transport-related protein NtTOL9a in plant cells. AVRcap1b targets NtTOL9a to inhibit the cell death response mediated by NLR proteins NRC2 and NRC3, thereby helping the pathogenic bacteria to successfully infect plants and achieve colonization and reproduction in the plant body. At the same time, by inhibiting the immune defense response of plants, the pathogenic bacteria are more likely to spread and cause diseases in the plant body, causing serious impact on the growth and development of plants, and eventually leading to reduced yield of crops and major losses to agricultural production. AVRcap1b has the following structural characteristics: ① specific amino acid sequence: like many proteins, AVRcap1b is composed of specific amino acids arranged in a certain order, and the types, number and arrangement order of these amino acids determine its unique physicochemical properties and biological functions. ② signal peptide domain: the signal peptide is generally located at the N-terminus of the protein, guiding the directional transport and positioning of the protein in the cell. For AVRcap1b, the signal peptide domain can guide it to be secreted from the Oomycetes cell to the outside of the cell, and then into the plant cell to play a role. ③ effector domain: the effector domain is the key site of AVRcap1b to exert its regulation on the physiological functions of plant cells, and by interacting with the target protein in the plant cell, it interferes with the physiological processes such as immune response of the plant, thereby helping the pathogenic bacteria to infect the plant.

[0003] The study of AVRcap1b is of great significance. First, it reveals the interaction mechanism between plants and pathogens: studying the interaction between AVRcap1b and intracellular proteins in plants helps to understand how the plant immune system recognizes and resists the invasion of pathogens, and how pathogens evolve corresponding effector proteins to evade the plant immune response, which is of great significance to understand the co-evolution relationship between plants and pathogens. Second, it provides a target for plant disease-resistant breeding: after clarifying the key role of AVRcap1b in the pathogenic process of pathogens, corresponding disease-resistant strategies can be designed, such as cultivating plant varieties that can specifically recognize and resist AVRcap1b through gene editing technology, improving plant resistance to related pathogens, reducing the use of chemical pesticides, and achieving sustainable development of agriculture. Third, structural complementarity: the effector domain of AVRcap1b has a specific spatial conformation, and the amino acid residues on its surface form a specific shape and charge distribution. The target protein NtTOL9a in the plant cell also has a complementary binding site, and the two can precisely match each other, like "lock and key", to form specific binding.

[0004] Since 1993, when Hamers et al. discovered heavy chain antibodies naturally lacking light chains in camel blood, single domain antibodies (sdAb) have gradually replaced other small antibodies and become a hot spot for the development of new antibody drugs. Single domain antibodies, also known as nanobodies, are usually only about 15 KDa, about one tenth the size of traditional antibodies. They have disulfide bonds inside and a large number of hydrophilic residues on the surface, and have strong resistance to heat and pH. The properties of sdAb lacking Fc segment and light chain enable it to recognize hidden epitopes or small epitopes that cannot be recognized by traditional antibodies, and avoid complement reactions. In addition, single domain antibodies also have many advantages such as high stability, low toxicity, strong solubility, easy target screening, easy direct expression in prokaryotic microorganisms, and good economic efficiency. Sequence homology analysis shows that the VHH germline gene sequence of camel sdAb is highly homologous to human VH3, but CDR1 and CDR3 are slightly longer than humans, and CDR3 protrudes outward in the tertiary structure, so it is speculated that it has higher antigen binding specificity and affinity.

[0005] Based on the excellent properties of nanobodies, the development of nanobodies against AVRcap1b is of great significance. At the basic research level, it can reveal the immune mechanisms related to AVRcap1b target: AVRcap1b is an effector protein from P. infestans, and the study of nanobodies against AVRcap1b can help to better understand the interaction mechanism between plants and pathogens, especially how pathogens interfere with the immune response of plants through effector proteins, and how the immune system of plants recognizes and resists the invasion of pathogens. Nanobodies have small molecular weight and simple structure, by studying the binding mode of AVRcap1b nanobodies and AVRcap1b protein, the structure and function relationship of AVRcap1b protein can be more clearly analyzed, and the key action sites and action modes of AVRcap1b in the pathogenic process of pathogens can be determined. At the disease diagnosis level, nanobodies have high specificity and affinity, AVRcap1b nanobodies can be used to develop high sensitivity and specificity detection methods to quickly and accurately detect the presence of P. infestans or its related antigens, which helps to diagnose plant diseases early and take preventive measures in time to reduce losses in agricultural production. AVRcap1b nanobodies can be combined with various detection technologies, such as enzyme-linked immunosorbent assay (ELISA), immunofluorescence technology, and immunochromatography technology, to provide powerful tools for on-site rapid detection and laboratory accurate diagnosis of plant diseases, and improve detection efficiency and accuracy. At the disease treatment level, the good cell penetration ability and tissue permeability of nanobodies enable them to effectively reach the lesion site, AVRcap1b nanobodies can be used as a targeted carrier to specifically deliver therapeutic drugs or other active substances to the infection site, achieving precise attack on pathogens and reducing damage to normal tissues, and improving treatment effect. At the same time, nanobodies can also regulate the immune response of the body through interaction with the immune system. AVRcap1b nanobodies may have the ability to activate or enhance the plant's own immune defense mechanism, helping plants better resist P. infestans infection, and providing ideas for the development of new plant immune treatment methods. At the drug development level, it can provide help for the design of new drugs and the establishment of drug screening models, and the research of AVRcap1b nanobodies lays a foundation for the development of new antifungal drugs based on nanobodies. By modifying and modifying nanobodies, such as coupling with antibiotic, antifungal peptide and other drug molecules, or designing nanobody fusion proteins with enzyme activity, cytotoxicity and other functions, it is expected to develop more efficient and less toxic drugs against P. infestans. Using the specific interaction between AVRcap1b nanobodies and pathogens, a high-throughput drug screening model can be established to quickly screen out small molecule compounds or biological agents that can inhibit the activity of AVRcap1b protein or block its interaction with host cells, accelerating the research and development process of new antifungal drugs.

[0006] There are many limitations in the development of existing traditional AVRcap1b antibodies. Firstly, the cost is high in production: the production process of traditional antibodies is complex, which requires the use of animal cell culture technology, has high requirements for production environment and equipment, and has long cell culture period and limited yield, resulting in a substantial increase in production cost, which is not conducive to large-scale production. The long production cycle makes it difficult to quickly meet market demand. It takes months or even longer to complete the whole process from immunizing animals, screening positive hybridoma cells, to obtaining a cell strain stably secreting antibodies, and then large-scale culture to produce antibodies. Secondly, in terms of antibody performance, the stability is limited: traditional antibodies are easily denatured and inactivated under high temperature, acid and alkali conditions, and their stability cannot meet the needs of some special application scenarios, such as complex physiological environment in vivo or long-term storage. Poor tissue penetration: the molecular weight of traditional antibodies is large, which makes it difficult to effectively penetrate physiological barriers such as biological membranes and blood-brain barriers, limiting the treatment effect on some deep tissue or special site pathogen infection. The affinity and specificity are not ideal: although screened and optimized, the affinity and specificity of traditional antibodies still have room for improvement, which may cross-react with other unrelated antigens, affecting the accuracy of detection and treatment. Thirdly, in terms of immunogenicity, it is easy to trigger immune response: traditional antibodies are mostly derived from animals and are exogenous proteins to the human body, which have strong immunogenicity and can easily cause immune response, produce anti-antibodies, not only reduce the treatment effect, but also can cause allergic reactions and even lead to treatment failure. SUMMARY

[0007] The present application aims at the above-mentioned problems, and provides an anti-AVRcap1b nanobody developed based on yeast two-hybrid technology and its application. The development cycle is short, and the antibody quality is high. The anti-AVRcap1b nanobody has outstanding advantages in terms of antibody affinity and targeting specificity, small molecule size, stability and transformability, production cost, etc.

[0008] The specific scheme adopted by the present application is as follows:

[0009] In a first aspect, the present application provides an AVRcap1b nanobody, which is AVRcap1b nanobody I, AVRcap1b nanobody II, AVRcap1b nanobody III or AVRcap1b nanobody IV.

[0010] The complementary binding region of the AVRcap1b nanobody I comprises CDR1, CDR2 and CDR3, wherein the CDR1 is an amino acid sequence as shown in SEQ ID NO: 2, the CDR2 is an amino acid sequence as shown in SEQ ID NO: 3, and the CDR3 is an amino acid sequence as shown in SEQ ID NO: 4.

[0011] The complementary binding region of the AVRcap1b Nanobody II comprises a CDR1 of an amino acid sequence as shown in SEQ ID NO: 7, a CDR2 of an amino acid sequence as shown in SEQ ID NO: 8, and a CDR3 of an amino acid sequence as shown in SEQ ID NO: 9.

[0012] The complementary binding region of the AVRcap1b Nanobody III comprises a CDR1 of an amino acid sequence as shown in SEQ ID NO: 12, a CDR2 of an amino acid sequence as shown in SEQ ID NO: 13, and a CDR3 of an amino acid sequence as shown in SEQ ID NO: 14.

[0013] The complementary binding region of the AVRcap1b Nanobody IV comprises a CDR1 of an amino acid sequence as shown in SEQ ID NO: 17, a CDR2 of an amino acid sequence as shown in SEQ ID NO: 18, and a CDR3 of an amino acid sequence as shown in SEQ ID NO: 19.

[0014] As a further optimization of the above-mentioned AVRcap1b Nanobody, the amino acid sequence of the AVRcap1b Nanobody I is shown in SEQ ID NO: 1; the amino acid sequence of the AVRcap1b Nanobody II is shown in SEQ ID NO: 6; the amino acid sequence of the AVRcap1b Nanobody III is shown in SEQ ID NO: 11; and the amino acid sequence of the AVRcap1b Nanobody IV is shown in SEQ ID NO: 16.

[0015] In a second aspect, the present application provides a nucleic acid molecule encoding the above-mentioned AVRcap1b Nanobody. Further, the nucleic acid molecule sequence encoding the AVRcap1b Nanobody I is shown in SEQ ID NO: 5; the nucleic acid molecule sequence encoding the AVRcap1b Nanobody II is shown in SEQ ID NO: 10; the nucleic acid molecule sequence encoding the AVRcap1b Nanobody III is shown in SEQ ID NO: 15; and the nucleic acid molecule sequence encoding the AVRcap1b Nanobody IV is shown in SEQ ID NO: 20.

[0016] In a third aspect, the present application provides a nucleic acid construct comprising the above-mentioned nucleic acid molecule. Further, the nucleic acid construct is a vector or a host cell.

[0017] In a fourth aspect, the present application provides the above-mentioned AVRcap1b Nanobody, nucleic acid molecule or nucleic acid construct for use in the study of the immune mechanism related to the AVRcap1b target.

[0018] In a fifth aspect, the present application provides the above-mentioned AVRcap1b Nanobody, nucleic acid molecule or nucleic acid construct for use in the development of a product for early diagnosis of plant diseases.

[0019] In a sixth aspect, the present application provides the use of the above-mentioned AVRcap1b nanobody, nucleic acid molecule or nucleic acid construct in the development of an anti-bacterial infection drug.

[0020] Beneficial effects: The present application uses a yeast two-hybrid screening system, the screening cost is greatly reduced compared to traditional antibodies, the screening period is shorter, the requirements for experimental environment and equipment are simpler, and the applicability is wide. The system studies protein interactions in living cells, can reflect the true interaction of proteins in the physiological state, is closer to the natural environment in vivo, and makes the research results more convincing. The use of high-copy and strong-promoter expression vectors, combined with sensitive detection methods such as yeast phenotype, X-gal and His3 protein expression, can detect weaker protein interactions. The use of multiple screening steps, such as reporter gene verification and double selection of yeast strains, can effectively reduce false positive results caused by non-specific interactions and improve the accuracy of screening results. Compared with traditional AVRcap1b antibodies, the AVRcap1b nanobody has smaller molecular weight, better tissue penetration, and is more stable and less immunogenic. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 is a self-activation detection result diagram.

[0022] Figure 2 is a library screening result diagram.

[0023] Figure 3 is a rotation verification result diagram; in the diagram, (+) is a positive control pGBKT7-p53+pGADT7-largeT, and (-) is a negative control pGBKT7-laminC+pGADT7-largeT. DETAILED DESCRIPTION

[0024] The present application uses the AVRcap1b gene constructed into the pGBKT7 vector as a bait to screen a yeast two-hybrid nanobody library. Through multiple reporter gene detection, DNA sequencing and BLAST alignment analysis of positive clones, the nanobody interacting with AVRcap1b is determined.

[0025] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the embodiments of the present application. Unless otherwise specified, the reagents or materials used below are all commercially available, and the methods used are all conventional technical means.

[0026] I. Transforming the bait plasmid into the recipient strain AH109 and detecting self-activation thereof

[0027] 1.1 Yeast transformation

[0028] The following plasmids were transformed into AH109 respectively:

[0029]

[0030] 1.2 Preparation of yeast competent cells and transformation method

[0031] 1. A single colony of AH109 was picked from YPDA plate and inoculated into 4ml YPDA liquid medium, 30℃, 225rpm, shaking culture for 18-20h (overnight) until OD 600 >1.5.

[0032] 2. The culture was transferred into 50ml YPDA liquid medium, with initial OD 600 =0.2, 30℃, 225rpm, shaking culture for 4-5h until OD 600 =0.6.

[0033] 3. The cells were collected by centrifugation at 4000rpm for 5min at room temperature.

[0034] 4. The cells were resuspended in 20ml sterile water, mixed well, and collected by centrifugation at 4000rpm for 5min at room temperature, and the supernatant was discarded.

[0035] 5. The cells were resuspended in 5ml 0.1M LiAc, mixed well, and collected by centrifugation at 4000rpm for 5min at room temperature, and the supernatant was discarded.

[0036] 6. The cells were resuspended in 500ul 0.1M LiAc, mixed well, and aliquoted into 1.5ml centrifuge tubes, 50ul per tube (for each transformation), and stored for use.

[0037] 7. The following reagents were added to each 1.5ml centrifuge tube in turn, mixed well with a gun tip or shaken vigorously for about 1min until completely mixed.

[0038]

[0039] 8. Incubate in a 30℃ water bath for 30min.

[0040] 9. Heat shock in a 42℃ water bath for 25min.

[0041] 10. Recover in a 30℃ water bath for 30min.

[0042] 11. The cells were collected by centrifugation at 4000rpm for 5min at room temperature, and the supernatant was discarded.

[0043] 12. The cells were resuspended in 200ul sterile water for each transformation, mixed as gently as possible, and plated on the corresponding deficient selection plate.

[0044] 13. Incubate at 30℃ for 4d.

[0045] 1.3 Auto-activation detection

[0046] 8 single colonies were randomly picked from the yeast transformants grown from pGBKT7-AVRcap1b+pGADT7 co-transformation of AH109 and preserved for PCR detection. 3 points were randomly picked from the single colonies with correct PCR detection results and inoculated on SD-TL, SD-TLH, SD-TLHA, SD-TLHA+X-α-gal plates and cultured at 30°C for 3-5 days.

[0047] From the experimental results, it can be seen that the positive control can grow on SD-TL, SD-TLH, SD-TLHA, SD-TLHA+X-α-gal plates and can color on SD-TLHA+X-α-gal plate. The negative control grows on SD-TL plate and does not grow on SD-TLH, SD-TLHA, SD-TLHA+X-α-gal plates. The experimental group and the negative control group grow consistently, indicating that pGBKT7-AVRcap1b+pGADT7 has no auto-activation phenomenon. The results are shown in the following table: Figure 1 .

[0048] II. Library screening

[0049] The AH109 yeast strain containing the correct pGBKT7-AVRcap1b bait plasmid was used as the acceptor to prepare the competent cells, and the library plasmid pGADT7-VHH was transformed into it, and the SD-TLH selection plate was coated.

[0050] 2.1 Library DNA transformation method:

[0051] 1. Single strain was picked from SD-T plate and inoculated in liquid SD-T medium 50ml, 30°C, 225rpm, and cultured for 24h.

[0052] 2. Inoculated in YPDA liquid 500ml, with initial OD 600 =0.2, 30°C, 225rpm, and cultured for 4-5h until OD 600 =0.6.

[0053] 3. Centrifuged to collect bacteria at room temperature, 4000rpm, 5min.

[0054] 4. Resuspend the bacterial cells with 30ml sterile water, mix well, centrifuge to collect bacteria at room temperature, 4000rpm, 5min, and discard the supernatant.

[0055] 5. Resuspend the bacterial cells with 20ml 0.1M LiAc, mix well, centrifuge to collect bacteria at room temperature, 4000rpm, 5min, and discard the supernatant.

[0056] 6. Resuspend the cells in 10 ml 0.1 M LiAc, mix well, centrifuge the cells, 4000 rpm, 5 min, at room temperature, discard the supernatant.

[0057] 7. Add the following reagents to the centrifuge tube in order, mix well by pipetting or vortex vigorously for 1 min or until well mixed.

[0058]

[0059] 8. Incubate at 30°C for 30 min.

[0060] 9. Heat shock at 42°C for 25 min.

[0061] 10. Recover at 30°C for 1 h.

[0062] 11. Centrifuge the cells, 4000 rpm, 5 min, at room temperature, discard the supernatant, resuspend the cells in 6 ml sterile water, mix gently, take 20 μl of the culture and dilute it, then spread it on SD-TL plates for detecting the transformation efficiency of the library. Spread the rest on SD-TLH plates, 20 plates in total.

[0063] 12. Incubate at 30°C for 3-7 days, observe the growth of the colonies.

[0064] 13. Pick the grown colonies and transfer them to SD-TLHA selection plates for further incubation for 3-5 days.

[0065] 2.2 Screening results

[0066] Positive yeast clones were obtained by screening on SD-TLHA selection plates, and colonies grew on the screening SD-TLHA plates, so 96 grown colonies were picked from the plates for PCR verification. The results are shown in the attached Figure 2 .

[0067] III. Identification and sequencing of positive yeast clones

[0068] In order to identify the positive clones screened on the SD-TLHA plates, the positive clones were amplified from the yeast cells for DNA sequencing, and BLAST analysis was performed by comparing the sequences with the sequences in the GenBank database.

[0069] Sequencing and alignment results: After PCR amplification and sequencing of the positive yeast clones, Seqman and BLAST alignment were performed, and finally 5 gene sequences were obtained.

[0070] IV. Verification of the positive yeast clones

[0071] The positive clones grown on the above-mentioned SD-TLHA-deficient plates were diluted with sterile water and then spotted on SD-TL, SD-TLH, SD-TLHA and SD-TLHA+X-α-gal deficient plates, and incubated at 30℃ for 3-4 days. Figure 3 The results of the rotation verification show that the positive control can grow on the SD-TL, SD-TLH, SD-TLHA and SD-TLHA+X-α-gal deficient plates, and show blue color on the SD-TLHA+X-α-gal deficient plate; the negative control can only grow on the SD-TL plate, but cannot grow on the other plates. The five yeast positive clones screened can all grow normally on the SD-TL deficient plate, and the four yeast positive clones screened can all grow normally on the SD-TLH, SD-TLHA and SD-TLHA+X-α-gal deficient plates and show blue color on the SD-TLHA+X-α-gal plate.

[0072] The present application is verified by the rotation experiment, and it is proved that the four yeast positive clones screened are all positive.

[0073] The present application successfully screens four AVRcap1b nanobodies by using the yeast two-hybrid screening system. The present application obtains the AVRcap1b nanobody gene in a short time by combining the use of the yeast two-hybrid technology, and effectively reduces the development and production cost of the AVRcap1b nanobody, which shows that the AVRcap1b nanobody obtained by the present application has a continued development value.

[0074] The four AVRcap1b nanobodies are AVRcap1b nanobody I, AVRcap1b nanobody II, AVRcap1b nanobody III and AVRcap1b nanobody IV, respectively.

[0075] The amino acid sequence of the AVRcap1b nanobody I is shown as SEQ ID NO: 1, the CDR1 is shown as SEQ ID NO: 2, the CDR2 is shown as SEQ ID NO: 3, and the CDR3 is shown as SEQ ID NO: 4. The nucleotide sequence is shown as SEQ ID NO: 5.

[0076] The amino acid sequence of the AVRcap1b nanobody II is shown as SEQ ID NO: 6, the CDR1 is shown as SEQ ID NO: 7, the CDR2 is shown as SEQ ID NO: 8, and the CDR3 is shown as SEQ ID NO: 9. The nucleotide sequence is shown as SEQ ID NO: 10.

[0077] The amino acid sequence of the AVRcap1b Nanobody IV is shown as SEQ ID NO: 16, with CDR1 shown as SEQ ID NO: 17, CDR2 shown as SEQ ID NO: 18, and CDR3 shown as SEQ ID NO: 19. The nucleotide sequence is shown as SEQ ID NO: 20.

[0078] The amino acid sequence of the AVRcap1b Nanobody IV is shown as SEQ ID NO: 16, with CDR1 shown as SEQ ID NO: 17, CDR2 shown as SEQ ID NO: 18, and CDR3 shown as SEQ ID NO: 19. The nucleotide sequence is shown as SEQ ID NO: 20.

[0079] The amino acid sequence of the AVRcap1b Nanobody IV is shown as SEQ ID NO: 16, with CDR1 shown as SEQ ID NO: 17, CDR2 shown as SEQ ID NO: 18, and CDR3 shown as SEQ ID NO: 19. The nucleotide sequence is shown as SEQ ID NO: 20.

[0080] The amino acid sequence of the AVRcap1b Nanobody IV is shown as SEQ ID NO: 16, with CDR1 shown as SEQ ID NO: 17, CDR2 shown as SEQ ID NO: 18, and CDR3 shown as SEQ ID NO: 19. The nucleotide sequence is shown as SEQ ID NO: 20.

[0081] QVQLQESGGGLVQAGGSLRLSCAASGTIFHHYNMGWYRQAPGKERELVASINDG GNTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAACRSQITQLPYWGQHPG HSQ

[0082] [FR1: QVQLQESGGGLVQAGGSLRLSCAAS; FR2: GWYRQAPGKER; FR3: ADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC; FR4: WGQGTQVTDSS; CDR1: GTIFHHYNMG; CDR2: ELVASINDGGNTYY; CDR3: ACRSQITQLPY]

[0083] The amino acid sequence of the AVRcap1b Nanobody IV is shown as SEQ ID NO: 16, with CDR1 shown as SEQ ID NO: 17, CDR2 shown as SEQ ID NO: 18, and CDR3 shown as SEQ ID NO: 19. The nucleotide sequence is shown as SEQ ID NO: 20.

[0084] QVQLQESGGGLVQAGGSLRLSCAASGTIFHHYNMGWYRQAPGKERELVASINDG GNTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAACRSQITQLPYWGQHPG HSQ

[0085] [FR1 : QVQLQESGGGLVQAGGSLRLSCAAS; FR2: GWYRQAPGKER; FR3: ADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC; FR4: WGQGTRSQS; CDR1 : GSIFGPLLM; CDR2: EFVASISSGGITYY; CDR3: AVTSYWHLLIARIHQY]

[0086] Amino acid sequence of AVRcap1 b Nanobody IV (SEQ ID NO: 16):

[0087] QVQLQESGGGLVQAGGSLRLSCAASGYISSPNLMGWYRQAPGKERELVAGISRGS STYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAPGTYCKFHWYWGQGTR VTVSS

[0088] [FR1 : QVQLQESGGGLVQAGGSLRLSCAAS; FR2: GWYRQAPGKER; FR3: ADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC; FR4: WGQGTRSQS; CDR1 : GSIFGPLLM; CDR2: EFVASISSGGITYY; CDR3: AVTSYWHLLIARIHQY]

[0089] Amino acid sequence of AVRcap1 b Nanobody IV (SEQ ID NO: 16):

[0090] QVQLQESGGGLVQAGGSLRLSCAASGYISSPNLMGWYRQAPGKERELVAGISRGS STYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAPGTYCKFHWYWGQGTR VTVSS

[0091] [FR1 : QVQLQESGGGLVQAGGSLRLSCAAS; FR2: GWYRQAPGKER; FR3: ADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC; FR4: WGQGTRSQS; CDR1 : GSIFGPLLM; CDR2: EFVASISSGGITYY; CDR3: AVTSYWHLLIARIHQY]

[0092] CDR1 : GYISSPNLM; CDR2: ELVAGISRGSSTYY; CDR3: AAPGTYCKFHWY]

[0093] The DNA sequence information of the four AVRcap1b nanobodies of the application is as follows:

[0094] The DNA sequence of AVRcap1b nanobody I (SEQ ID NO: 5):

[0095] CAGGTTCAACTACAGGAGTCAGGCGGGGGGCTCGTACAAGCCGGTGGAAGCTT ACGGCTTTCTTGTGCTGCATCCGGCTCTATATTTTGTTCTTGTTTTATGGGTTGGTATAGGCAAGCGCCGGGAAAAGAACGCGAATTTGTGGCAGGTATCAATTTTGGAGGTAGTACCTATTACGCCGATTCTGTAAAGGGTCGTTTCACAATTTCGCGAGACAATGCAAAAAACACTGTCTATCTGCAGATGAACAGCTTGAAGCCTGAGGATACAGCGGTTTACTACTGCGCCGTTACGTTGAGGGCGACGACTACTCATGATTATTGGGGGCAAGGCACCCAGGTCACAGACAGTAGT

[0096] The DNA sequence of AVRcap1b nanobody II (SEQ ID NO: 10):

[0097] CAGGTTCAACTACAGGAGTCAGGCGGGGGGCTCGTACAAGCCGGTGGAAGCTT ACGGCTTTCTTGTGCTGCATCCGGCACTATATTTCATCATTATAATATGGGTTGGTATAGGCAAGCGCCGGGAAAAGAACGCGAACTTGTGGCAAGTATCAATGATGGAGGTAATACCTATTACGCCGATTCTGTAAAGGGTCGTTTCACAATTTCGCGAGACAATGCAAAAAACACTGTCTATCTGCAGATGAACAGCTTGAAGCCTGAGGATACAGCGGTTTACTACTGC GCCGCTTGTAGGT CGCAGATTACTCAGCTTCCGTATTGGGGGCAGCACCCAGGTCACAGTCAG

[0098] The DNA sequence of AVRcap1b nanobody III (SEQ ID NO: 15):

[0099] CAGGTTCAACTACAGGAGTCAGGCGGGGGGCTCGTACAAGCCGGTGGAAGCTT ACGGCTTTCTTGTGCTGCATCCGGCTATATATCTTCGCCTAATCTGATGGGTTGGTATAGGCAAGCGCCGGGAAAAGAACGCGAACTTGTGGCAGGTATCAGTCGGGGAAGTAGTACCTATTACGCCGATTCTGTAAAGGGTCGTTTCACAATTTCGCGAGACAATGCAAAAAACACTGTCTATCTGCAGATGAACAGCTTGAAGCCTGAGGATACAGCGGTTTACTACTGCGCCGCTCCGGGGACGTATTGTAAGTTTCATTGGTATTGGGGGCAAGGCACCCGGGTCACAGTCAGTAGT

[0100] DNA sequence of the AVRcap1b Nanobody IV (SEQ ID NO: 20):

[0101] CAGGTTCAACTACAGGAGTCAGGCGGGGGGCTCGTACAAGCCGGTGGAAGCTT ACGGCTTTCTTGTGCTGCATCCGGCTATATATCTTCGCCTAATCTGATGGGTTGGTATAGGCAAGCGCCGGGAAAAGAACGCGAACTTGTGGCAGGTATCAGTCGGGGAAGTAGTACCTATTACGCCGATTCTGTAAAGGGTCGTTTCACAATTTCGCGAGACAATGCAAAAAACACTGTCTATCTGCAGATGAACAGCTTGAAGCCTGAGGATACAGCGGTTTACTACTGCGCCGCTCCGGGGACGTATTGTAAGTTTCATTGGTATTGGGGGCAAGGCACCCGGGTCACAGTCAGTAGT

[0102] It should be noted that the above-mentioned embodiments are understood as illustrative rather than limiting the scope of protection of the present application, which is defined by the claims. Some non-essential improvements and adjustments to the present application, without departing from the spirit and scope of the present application, are still within the scope of protection of the present application for those skilled in the art.

Claims

1. An AVRcap1 b Nanobody, characterized in that: The AVRcap1b Nanobody is an AVRcap1b Nanobody I; The complementary binding region of the AVRcap1b Nanobody I comprises a CDR1 of the amino acid sequence as shown in SEQ ID NO: 2, a CDR2 of the amino acid sequence as shown in SEQ ID NO: 3, and a CDR3 of the amino acid sequence as shown in SEQ ID NO:

4.

2. The AVRcap1 b Nanobody according to claim 1, characterized in that: The amino acid sequence of the AVRcap1b Nanobody I is as shown in SEQ ID NO:

1.

3. A nucleic acid molecule, characterized in that: The nucleic acid molecule encodes the AVRcap1b Nanobody as claimed in claim 2.

4. The nucleic acid molecule of claim 3, wherein: The nucleic acid molecule sequence encoding the AVRcap1b Nanobody I is as shown in SEQ ID NO:

5.

5. A nucleic acid construct, characterized in that: The nucleic acid molecule as claimed in claim 4.

6. The nucleic acid construct of claim 5, wherein: The nucleic acid construct is a vector or a host cell.

7. Use of the AVRcap1b Nanobody according to any one of claims 1-2, the nucleic acid molecule according to any one of claims 3-4, or the nucleic acid construct according to any one of claims 5-6, in the development of a product for early diagnosis of a plant disease, the plant disease being potato late blight.

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